Increased body mass index is linked to systemic inflammation through altered chromatin co-accessibility in human preadipocytes

Obesity-induced adipose tissue dysfunction can cause low-grade inflammation and downstream obesity comorbidities. Although preadipocytes may contribute to this pro-inflammatory environment, the underlying mechanisms are unclear. We used human primary preadipocytes from body mass index (BMI) -discordant monozygotic (MZ) twin pairs to generate epigenetic (ATAC-sequence) and transcriptomic (RNA-sequence) data for testing whether increased BMI alters the subnuclear compartmentalization of open chromatin in the twins’ preadipocytes, causing downstream inflammation. Here we show that the co-accessibility of open chromatin, i.e. compartmentalization of chromatin activity, is altered in the higher vs lower BMI MZ siblings for a large subset ( ~ 88.5 Mb) of the active subnuclear compartments. Using the UK Biobank we show that variants within these regions contribute to systemic inflammation through interactions with BMI on C-reactive protein. In summary, open chromatin co-accessibility in human preadipocytes is disrupted among the higher BMI siblings, suggesting a mechanism how obesity may lead to inflammation via gene-environment interactions.

The NEAT v1.2.3 R package was used to test for GO term enrichment in the A compartment clusters. To provide network information to NEAT, we used the WGCNA v1.72 R package. The online tool REVIGO was used to cluster GO terms based on semantic similarity. Transcription factor motif enrichment in the ATAC-seq peaks was performed using HOMER v4.11.1. KEGG pathway enrichment analysis was performed using the online tool WebGestalt.
Differential accessibility between PAd and differentiating PAd (D1) ATAC-seq data was done using the limma v3.34.9 R package. Differential correlation analysis was done using the cocor v1.1 R package.
To estimate partitioned heritability for BMI and obesity-related traits, partitioned LD score regression (LDSC v1.0.1) was used. For genotypeby-environment interaction testing, we used plink v1.90b3.45. The publicly available software MAGENTA v2.4 was used to test for regional GWAS SNP enrichments.
All other statistical analyses were performed using R v3.6.3.
Both the raw counts and normalized counts in transcript per million (TPMs) of the RNA-seq and ATAC-seq data used in the analyses of this study are available at the GEO database under the accession number GSE235363 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235363], and source data are provided with this paper. The data that support the GxE findings in this manuscript were generated using the UK Biobank under the UK Biobank Application Number 33934. These data are available under restricted access for bona fide researchers through the application process. The round 2 UK Biobank GWAS summary statistics used in this study are publicly available [http://www.nealelab.is/uk-biobank/]. The human reference genome (1000 Genomes human_g1k_v37) used in this study is available at IGSR [https://www.internationalgenome.org/category/assembly/]. The PAd pCHi-C data are available at the GEO database under the accession number GSE183770 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE183770]. The ChIP-seq data for the H3K27ac histone mark and MED1 at the day 1 adipogenic time point from bone marrow derived stromal stem cells (BM-hMSC-TERT4) are available at the GEO database under the accession number GSE113253 [https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE113253]. The ENCODE blacklist used in this study is available at the ENCODE portal under the accession number ENCFF001TD [https://www.encodeproject.org/annotations/ENCSR636HFF/]. All data supporting the findings described in this manuscript are available in the article and in the Supplementary Information and from the corresponding author upon request.
We report the nearly balanced design between male and female sex in our BMI-discordant MZ twin study and correct for sex in our ATAC-seq and RNA-seq data. We did not perform sex-based analyses due to the limited sample size in the current study.
Only age and sex were controlled for in our analyses. No socially constructed or socially relevant categorization variables were used in our analyses.
The BMI-discordant MZ twin pairs were identified from the population-based Finnish Twin Cohorts (FTC), and selected based on large intrapair differences in BMI (>=3 kg/m2). The mean age of the BMI-discordant pairs is 47 years (+/-2y (s.e.)) and comprises 22 pairs of males (42%) and 31 pairs of females (58%). The subset of twins from the ongoing Finnish twin cohorts (FTC) for which we cultured and collected preadipocyte data has a mean age of 42 y (+/-4 y (s.e.)), and it comprises 6 pairs of males (60%) and 4 pairs of females (40%).
The GxE research was conducted using the UK Biobank Resource. The UK Biobank consists of~500,000 individuals with genotypes and phenotypes and includes males and females from a broad range of ages. The maximum number of individuals was included to increase the power of discover of GxE interactions (n=up to 372,652 non-related individuals). This population sample has a mean age of 57 y +/-8 y (s.d.) and comprises 54% males and 46% females.

April 2023
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All studies must disclose on these points even when the disclosure is negative. For the ATAC-seq and RNA-seq data collection from the BMI-discordant monozygotic (MZ) twin pairs' preadipocytes (ATAC + RNA) and differentiating preadipocytes (day 1 of differentiation, ATAC only), sample size was determined by the successful culture of the preadipocytes for 10 pairs (n=20). The gene-environment interaction (GxE) analysis was conducted using the UK Biobank Resource. The UK Biobank consists of~500,000 individuals with genotypes and phenotypes. The maximum number of individuals available was included to increase the power of discover of GxE interactions (n=up to 372,652 unrelated Europeans). The used sample sizes are typical for published functional genomics experiments in ENCODE and other functional databases, which are limited by the availability of human biopsies and primary cell biobanking. The functional priors established through our experiments helped identify regions with altered open chromatin co-accessibility harboring variants that contribute to systemic inflammation through their interactions with BMI on C-reactive protein (CRP) in the more powerful UK Biobank analyses, further supporting the adequate sample sizes of the primary cell data.
For the ATAC-seq data, exclusion criteria related to data quality control (QC) were set ahead of time to correspond to ENCODE Data Standards. This resulted in the exclusion of 6 samples due to the following reasons: improper fragment size distribution (1); poor library complexity (4); and too few sequencing reads (1). To reduce spurious associations due to population substructure and genetic heterogeneity in the UK Biobank analysis, related and non-Caucasian individuals were excluded.
Two BMI-discordant MZ twin pairs were cultured in two independent cultures to serve as replication to test for the reproducibility of the ATAC-seq data. We collected PAd (n=4) and differentiating PAd (n=4) ATAC-seq data for these two pairs. Seven of the eight isogenic biological replicates from the same individual were retained after QC. These samples were used to assess the reproducibility of the data: the uncorrected peak TPMs for the seven isogenic biological replicates were correlated at a mean Spearman's rho of 0.96. For comparison, the inter-individual PAd samples were correlated at a mean Spearman's rho of 0.87 and the inter-individual differentiating PAd samples were correlated at a mean Spearman's rho of 0.83.
Results reported from the BMI-discordant MZ twin cohort were not replicated due to the unique nature of the cohort in identifying epigenetic differences across BMI levels within MZ pairs. Results reported from the UK Biobank GxE analyses were not replicated due to the unprecedented large sample size of the cohort and because individual GxE SNPs were not identified after correction for multiple testing.
N/A. This is an observational study, so no randomization was performed.
N/A. This is an observational study, so no randomization was performed.